The inventors of the present invention have investigated relationship between cancer growth or its suppression and activity of heme oxygenase, and found that heme oxygenase is highly expressed in tumor tissues. The heme oxygenase degrades heme and produces bilverdin, carbon monoxide and free iron in tumor or normal tissues.
Bilverdin is readily converted into bilirubin in the cells, and this bilirubin is a very potent antioxidant. Thereby, bilirubin can be a defense molecule against active oxygen such as superoxide, H2O2, or nitric oxide etc that are generated by leukocytes of the hosts (cancer patients). Namely, bilirubin, thus generated will nullify the toxic oxidative defense power against cancer cells or infecting microbes of the host. Therefore, if one blocks heme oxygenase, no bilirubin will be available and tumor cells will be killed by the oxidative molecules generated by leukocytes as a result of innate defense state.
The inventors had tried to see the antitumor effect of zinc protoporphyrin (ZnPP), an inhibitor of heme oxygenase, administered into the tumor feeding artery of tumor bearing rats thereby targeting the inhibitor into the tumor loci selectively, and they indeed confirmed antitumor effect in rats (K. Doi et al.: Br. J. Cancer 80, 1945–54, 1999).
However, there are several problems to use ZnPP per se as an antitumor agent. First, it is almost insoluble in water per se, thus, we had to use oily formulation to solubilize ZnPP, and such oily formulated agent may be only injectable via the tumor-feeding artery, and this is rather too elaborate and far advanced skill is required for this procedure compared with ordinary intravenous or subcutaneous injection. Second, native or original ZnPP has no guarantee for selective accumulation of ZnPP in cancer tissues, and to exert tumor selective anticancer effect, whereas the drug will be widely distributed to whole body besides tumor. Therefore, unexpected side effects are concerned.
On the contrary, the inventors are experts in tumor biology, particularly study on the vascular permeability of solid tumor tissues, and knowledgeable that macromolecular therapeutics would permeate more selectively at the tumor tissue by virtue of unique anatomical character and by the effect of multiple vascular permeability factors; and further, those macromolecules are retained in the tumor tissues for long period. Thus, this phenomenon was coined “enhanced permeability and retention (EPR)-effect” (Y. Matsumura, H. Maeda: Cancer Res. 47: 6387–92, 1986; H. Maeda: In Advances in Enzyme Regulation (by G. Weber ed), Elsevier Scientific Ltd., Amsterdam, 41, 189–207, 2001).
According to the EPR-effect, drugs with molecular size larger than 40,000 exhibit high concentration in blood plasma for prolonged time, and several hours to days after intravenous injection; whereas intratumoral concentration will result in several fold higher more precisely in 24–48 hr time. This means, making the apparent drug size greater than 40,000, would make it possible for selective tumor targeting of such macromolecular drugs.
Meantime, various metal porphyrin derivatives having inhibitory activity against heme oxygenase, and improved method of their administrations as a whole were studied. The resulted finding is that amphipathic or water soluble polymer conjugation to the metal protporphyrins made it possible to yield water soluble metal porphyrin derivatives and they can be administered not only arterially but also intravenously which has more versatile and easy clinical usage. They exhibited EPR-effect by polymer conjugation yielding highly efficient accumulation in tumor, and enzyme inhibitory activity against heme oxygenase is retained for long period. As a result, only 2 to 3 times of injections made it possible to suppress tumor growth completely in mice, which was a remarkable result.
Previously, metal porphyrin derivatives possessing heme oxygenase inhibitory activity with amphipathic or water soluble polymer conjugation were never reported, nor were the method of their preparation before our own. Present inventors have developed the method for synthesis of amphipathic or water-soluble polymer conjugation of metalloporphyrin via amide linkage. Resultant polymer conjugated metalloporphyrin derivatives are novel series of compounds not reported previously.